CN106581761B - A kind of artificial liver tissue and preparation method thereof - Google Patents

Abstract

A kind of artificial liver tissue and preparation method thereof, the artificial liver organization includes the lower die stacked gradually from bottom to top, first microporous barrier, intermediate chip, second microporous barrier and upper die, in lower die, it is respectively formed down on intermediate chip and upper die, in, upper three layers of chamber, the culture solution containing growth factor is perfused respectively in three layers of chamber, extracellular matrix containing hepatic parenchymal cells and the suspension containing endothelial cell, it is isolated between adjacent chamber by microporous barrier, microporous barrier can obstruct travel motion of the flowing of extracellular matrix without influencing cell, by upper, lower chambers go out, entrance feed flow is simultaneously cultivated, endothelial cell passes through the extracellular matrix of intermediate cavity and forms lumen under the induction of growth factor, form the artificial liver tissue containing capillary.The artificial liver organization can bionical human body well liver organization, the functions such as mass transfer, selective penetrated property, toxic reaction of simulated liver internal capillary blood vessel.

Description

A kind of artificial liver tissue and preparation method thereof

Technical field

The present invention relates to field of tissue engineering technology, more particularly to a kind of artificial liver tissue and preparation method thereof.

Background technique

Currently, due to lacking enough source of people donors, and the liver organization of animal origin and the liver of source of people have compared with Big difference, in order to meet human liver pathological study and drug test screening source demand, using human archeocyte construct outside Hepatic model has become a kind of mode being widely recognized as.

Micro-fluidic chip due to its with small in size, consumption less, integrated level is high, controllability is good and can construct complicated knot The advantages such as structure become one of the preferred embodiment of the outer hepatic model of construct.Common micro-fluidic is poly dimethyl silicon Oxygen alkane (PDMS), with good biocompatibility, physical and chemical, biochemical stability, translucency and oxygen permeability make it easy to construct Vitro hepatic model is also beneficial to observation of the later period to liver model.

Existing Vitro hepatic model is all constructed using only hepatic parenchymal cells mostly, rare various kinds of cell co-culture model Also there is no the capillary structures and function inside reduction liver.Since the capillary in liver includes substance in liver The multiple functions such as transmitting, selective penetrated property, the screening of macromolecular poisonous substance and toxic reaction, for lacking the model of capillary, nothing By being screened for the pathological study of liver or for drug test, all there is a problem of inaccuracy.

Summary of the invention

It is a primary object of the present invention to overcome the deficiencies of the prior art and provide a kind of artificial liver tissue and its production side Method.

To achieve the above object, the invention adopts the following technical scheme:

A kind of artificial liver tissue, including micro-fluidic chip, the micro-fluidic chip are provided with five-layer structure, including under Lower die, the first microporous barrier, intermediate chip, the second microporous barrier and the upper die stacked gradually on and, in the lower section core Be respectively formed down on piece, the intermediate chip and the upper die, in, upper three layers of chamber, in three layers of chamber from bottom to top Culture solution of the distribution perfusion containing growth factor, the extracellular matrix containing hepatic parenchymal cells and the suspension containing endothelial cell, adjacent chambers It is isolated by first microporous barrier with second microporous barrier between room, the microporous barrier can obstruct the stream of extracellular matrix The dynamic travel motion without influencing cell passes through the entry and exit feed flow of upper and lower chamber and cultivates, under the induction of growth factor Endothelial cell passes through the extracellular matrix of intermediate cavity and forms lumen, forms the artificial liver tissue containing capillary.

Further:

The lower die, the intermediate chip and the upper die are PDMS chip, are bonded by oxygen plasma Mode assembles, and the microporous barrier is clamped between the PDMS chip by the deformation of itself and the PDMS chip.

The microporous barrier is that collagen is fabricated to thickness in the film of 0.1 ± 0.05mm range, has aperture 0.03 ± 0.01mm range, hole distance of shaft centers 0.06 ± 0.02mm range microwell array.

In the entrance integration testing module of an at least chamber, detection module is integrated in the outlet of an at least chamber, for disease Reason research and drug test, it is preferable that the test module is the chip of the culture solution containing virus, toxin or drug, the inspection Survey the expression quantity detection module that module is liver Activity determination module, metabolic integrity detection module or functional product.

The length × width × height of the lower die, the intermediate chip and the upper die is 25mm × 25mm × 1mm； The length × width × height of lower chambers is 5mm × 5mm × 0.2mm, and the hole heart of chamber entrance is away from for 18mm；The length of intermediate cavity × wide × a height of 5mm × 5mm × 1mm, the hole heart of chamber entrance is away from for 15mm, chamber of the intermediate chip in the lower die Entrance corresponding position is equipped with through-hole；The length × width × height of upper chamber is 5mm × 5mm × 0.2mm, the hole heart of chamber entrance away from For 12mm, the upper chip is equipped with through-hole in the chamber entrance corresponding position of the intermediate chip, the lower die；Each chamber The aperture of room entrance is 1.5mm, is connected by 0.4mm wide between each chamber entrance and chamber and with the contour channel of chamber It is logical；Preferably, all vertical edges in each chamber all pour out the fillet that radius is 1mm, to avoid turbulent flow and bubble is formed；It is described Length × width x thickness of microporous barrier is 10mm × 10mm × 0.1mm.

It is preferred that using at least one of following configuration: the hepatic parenchymal cells is the primary hepatic parenchymal cells of people or people The cell line HepG2 of hepatic parenchymal cells；The extracellular matrix is gelatinous rat-tail I-type collagen or fibrin, pig Skin I-type collagen；The growth factor is combination any one or more in VEGF, EGF and FGF, the basic culture solution For the combination of DMEM+RPMI or the combination of ECM+DMEM, preferably each 50%；The endothelial cell is HUVECs or HMEC-1.

A kind of production method making the artificial liver tissue, comprising: make 5 layers of single layer of the micro-fluidic chip Structure；Using the lower surface of lower die described in oxygen plasma sputter process and the upper surface of the intermediate chip, in described Between chip lower surface on pad upper first microporous barrier it made to cover chamber without covering entrance, then pass through entrance pair The neat lower die and the intermediate chip simultaneously make the lower surface of the lower die and the upper surface button of the intermediate chip It closes, first microporous barrier is clamped wherein；Using identical operation fasten the intermediate chip upper surface and it is described on The lower surface of square chip, and second microporous barrier is clamped wherein；Hereafter the micro-fluidic chip fastened is placed on oven Middle baking keeps its bonding close.

Further:

The microporous barrier is the film for being fabricated to biocompatibility by the method for electrostatic spinning using collagen, is reused sharp The method of light punching processes microwell array on film.

The extracellular matrix for being uniformly mixed with hepatic parenchymal cells is poured into from the entrance of the intermediate cavity to the intermediate cavity, It is filled until in the duct of the outlet of the intermediate cavity, contains growth from the entrance of the lower chambers to lower chambers injection thereafter The culture solution of the factor injects endothelial cell suspension into the upper chamber from the entrance of the upper chamber, until the upper chamber Outlet duct in fill, standing a period of time makes endothelial cells sediment and adherent growth on microporous barrier, hereafter from described The entrance of upper chamber is continuously injected into culture solution to the upper chamber；Under the driving of growth factor, endothelial cell vascularization is simultaneously erected The extracellular matrix layer of the intermediate cavity is penetrated to migration, the supply for the factor that hereafter stops growing is changed to be continuously injected into culture Liquid finally forms the artificial liver tissue containing capillary in the intermediate cavity.

A method of using the artificial liver tissue, comprising: to the entrance of chamber injection virus, toxin or to Drug is detected, infusion is transmitted and spread into artificial liver tissue by the blood vessel of forming, and generates corresponding reaction；Pass through The detection module in the exit of chamber is detected, and liver Activity determination, metabolic integrity detection and functional production are preferably included The expression quantity of object detects.

Beneficial effects of the present invention:

The present invention provides a kind of artificial liver tissue containing orientation capillary, and the tissue is due to including functional blood Tubing, can preferably bionical human body liver organization, mass transfer, selective penetrated property, the poison of simulated liver internal capillary blood vessel Property reaction etc. functions, it can be made more accurate when being used for the pathological study of liver and drug toxicity test screen.Simultaneously as Hepatic tissue constructs on micro-fluidic chip, can easily integrate other processing and detection module, so that after hepatic tissue forming, Realize the research and detection in later period with can be convenient.

Compared with prior art, the invention has the following advantages that

Artificial liver tissue construction of the invention in can be convenient on micro-fluidic chip with environmental control module and later period Detection module is integrated, convenient for the various aspects application of this artificial liver organization；The microporous barrier used can be combined by electrostatic spinning to swash Light punching production realizes good biocompatibility and biochemical, physical and chemical stability by the material selection of electrostatic spinning process, no Damage can be generated to tissue and be conducive to cell attachment and migration to accelerate the vascularization in hepatic tissue；The artificial liver constructed Dirty tissue has functional capillaries, is capable of the liver organization of better bionical human body, simulated liver internal capillary blood vessel The functions such as mass transfer, selective penetrated property, toxic reaction, so that its application in the fields such as pathological study and drug screening detection obtains More accurate result.

Detailed description of the invention

Fig. 1 is each schematic diagram of a layer structure of chip of artificial liver of embodiment of the present invention tissue.

Fig. 2 is the structural schematic diagram that the chip of the embodiment of the present invention is completed.

Fig. 3 is the sectional view of three layers of mutually isolated chamber of the embodiment of the present invention.

Fig. 4 is the diagrammatic cross-section for the artificial liver tissue containing capillary that the embodiment of the present invention finally constructs.

Specific embodiment

It elaborates below to embodiments of the present invention.It is emphasized that following the description is only exemplary, The range and its application being not intended to be limiting of the invention.

Refering to fig. 1 to Fig. 4, in one embodiment, a kind of artificial liver tissue, including micro-fluidic chip, micro-fluidic core Piece has five-layer structure, is followed successively by the PDMS chip 1 of perfusion growth factor from bottom to top, microporous barrier 2, hepatic parenchymal cells is perfused PDMS chip 3, microporous barrier 4, the PDMS chip 5 that endothelial cell is perfused complete PDMS chip by the technique that oxygen plasma is bonded Bonding assembling, microporous barrier then passes through it and the deformation of PDMS is clamped between PDMS chip, thus from bottom to top formed three layers The chamber being isolated by microporous barrier；Culture solution of the distribution injection containing growth factor in three layers of chamber from the bottom up contains liver parenchyma The extracellular matrix and endothelial cell suspension of cell pass through the entry and exit feed flow of upper and lower chamber and cultivate, in growth factor It induces lower endothelial cell to pass through the extracellular matrix of intermediate cavity and form lumen, forms the artificial liver group containing capillary It knits.In entrance integration testing module, integrated detection module is being exported, the model can be realized in pathological study and drug test Etc. concrete application.

In a preferred embodiment, the microporous barrier of PDMS chip, electrostatic spinning combination laser boring production can be used to carry out structure Micro-fluidic chip is built, by penetrating microporous barrier by growth factor-induced endothelial cell on chip and containing hepatic parenchymal cells Blood vessel is sprouted in extracellular matrix, to construct the artificial liver tissue containing orientation capillary.

In a specific embodiment, Fig. 1 show 5 layers of structure of chip, wherein the external ruler of PDMS chip 1,3,5 Very little is 25mm × 25mm × 1mm length × width × height, and it is the fillet of 1mm that all vertical edges of chamber, which have radius, enters and leaves oral pore Diameter is 1.5mm, and all there are through-holes to avoid core below closing for the entrance corresponding position of all upper dies chip under it The entrance of piece, the width in channel are 0.4mm, and the method that the PDMS chip containing chamber uses standard soft lithographic makes, and are made It is punched with punch；Chamber size on chip 1 is 5mm × 5mm × 0.2mm length × width × height, and entrance pitch of holes is 18mm；Chamber size on chip 3 is 5mm × 5mm × 1mm length × width × height, and entrance pitch of holes is 15mm；On chip 5 Chamber size is 5mm × 5mm × 0.2mm length × width × height, and entrance pitch of holes is 15mm；The outside of microporous barrier 2 and microporous barrier 4 Having a size of 10mm × 10mm × 0.1mm long × width x thickness, is made using rat-tail I-type collagen by electrostatic spinning, then made The microwell array that aperture is 0.03mm, hole distance of shaft centers is 0.06mm is processed with laser boring.The mode of assembling is: producing After 5 layers of single layer structure of chip, the lower surface of oxygen plasma sputter process chip 1 and the upper surface of chip 3,3~5min are used After take out, upper microporous barrier 2 is padded on the lower surface of chip 3 it is made to cover chamber and enter and leave oral pore without covering, then in microscope The upper surface fastening of the lower lower surface and chip 3 for being aligned chip 1 and 3 by discrepancy oral pore and making chip 1, microporous barrier 2 is clamped Wherein；The upper surface of chip 3 and the lower surface of chip 5 are fastened using identical operation, and microporous barrier 4 is clamped wherein； Hereafter the micro-fluidic chip fastened is placed on makes to bond even closer for 80 DEG C of oven for baking 0.5 hour or so, can be completed The assembling of chip.

The microporous barrier it is preferable to use material be rat-tail I-type collagen, be fabricated to by the method for electrostatic spinning The film of 0.1mm is cut into 10mm × 10mm long × wide rear method using laser boring and processes aperture on it and be 0.03mm, the microwell array that hole distance of shaft centers is 0.06mm, so that microporous barrier can obstruct the flowing of extracellular matrix without influencing The travel motion of cell.

The microporous barrier is made using electrostatic spinning and the process of laser boring, preferred process are as follows: first uses glue The former method by electrostatic spinning makes the film for having biocompatibility that film thickness is about 0.1mm, is cut into 10mm × 10mm Square sheet, process the through-hole of intensive, aperture about 0.03mm on film using the method for laser boring later, this film thickness Can guarantee microporous barrier will not Human Umbilical Vein Endothelial Cells migration generate it is excessive influence and enough intensity is provided, this aperture can guarantee Microporous barrier can form transmembrane movement of the barrier without obstructing endothelial cell to the extracellular matrix of intermediate channel.

It is respectively the chip completion assembling that the artificial liver tissue construction process of the embodiment of the present invention uses shown in Fig. 2-Fig. 3 Overall structure diagram and chamber cross-sectional schematic diagram afterwards, carry out high-temperature sterilization after being completed, and hereafter use micro-fluidic chip The concrete operation step for constructing the artificial liver tissue containing capillary includes: from the entrance 6 of intermediate cavity to intermediate cavity 13 The extracellular matrix for being uniformly mixed with hepatic parenchymal cells is poured into, stops perfusion after filling in the duct of intermediate cavity outlet 9, Continuously injected afterwards from cavity of resorption chamber inlet 8 to lower chambers using micro-injection pump or air pump the culture solution containing growth factor and Waste liquid is collected in lower chambers outlet 11, and endothelial cell suspension is injected into upper chamber 14 from epicoele chamber inlet 7, until upper chamber exports Stop perfusion after filling in 10 duct, standing 12 hours makes endothelial cells sediment and adherent growth is on microporous barrier 4, hereafter opens Begin to be continuously injected into culture solution from epicoele chamber inlet 7 to upper chamber 14 using micro-injection pump or air pump and exports 10 in upper chamber Collect waste liquid in place；It is primary every observation in 6 hours later, between 3~6 days, under the driving of growth factor, endothelial cell blood vessel Change the extracellular matrix layer that simultaneously vertical migration penetrates intermediate cavity 13, the supply for the factor that stops growing at this time is changed to be continuously injected into training Nutrient solution then forms the artificial liver tissue containing capillary in intermediate cavity.

Preferred hepatic parenchymal cells are the primary hepatic parenchymal cells of people, and preferred extracellular matrix is gelatinous rat-tail I Collagen type；Preferred growth factor is the combination of VEGF, EGF and FGF, and the ingredient of the growth factor may include but not Blood can be promoted by being limited to vascular endothelial growth factor VEGF, fibroblast growth factor FGF, epithelical cell growth factor EGF etc. The growth factor that pipe generates；Preferred basic culture solution uses the combination of DMEM+RPMI each 50%；Preferred endothelial cell For HUVECs.Liver cell can also be cell line HepG2 of the hepatic parenchymal cells of people etc., and extracellular matrix can also be fiber A variety of collagens such as albumen, pigskin I-type collagen, basic culture solution can also be a variety of culture mediums such as ECM+DMEM, interior Chrotoplast can also be HMEC-1 etc..

Fig. 4 show the schematic diagram of the section structure for the artificial liver tissue containing capillary that building is completed, endothelial cell 15 are attached on microporous barrier 4 in upper chamber 14, under the induction of growth factor migrate and around microporous barrier 4 imperforate section 19, Across micropore 20, being uniformly mixed in the extracellular matrix 18 of hepatic parenchymal cells 16 for intermediate cavity 13 is entered, and close to micropore Film 4 forms endodermis again, and after growth factor successive induction, endothelial cell agglomerate migration in matrix 18 squeezes hepatic parenchymal cells 16, and self-organizing forms pipeline, as containing the artificial liver tissue of capillary 17.

After hepatic tissue molding, virus, toxin or drug chip module can be integrated before entrance, and inspection is integrated behind outlet Module is surveyed, injects virus, toxin or drug to be detected into entrance 7,8 by the chip module before entrance, and pass through outlet 10, the detection module at 11 is detected, can be realized this artificial liver organization pathological study and in terms of Concrete application.

The entrance test module can include but is not limited to the chip of the culture solution containing virus, poisonous substance, drug etc., note It transmits and spreads into hepatic tissue by the blood vessel of forming after entering upper and lower chamber, and generate corresponding reaction.The outlet is surveyed Die trial block can include but is not limited to the expression quantity of liver Activity determination module, metabolic integrity detection module and functional product Detection module etc..

To sum up, the present invention penetrates microporous barrier by growth factor-induced endothelial cell on micro-fluidic chip and real containing liver Blood vessel is sprouted in the extracellular matrix of cell plastid, generates the artificial liver tissue containing orientation capillary, the tissue is due to packet Containing functional vascular tissue, can better bionical human body liver organization, the mass transfer of simulated liver internal capillary blood vessel, choosing Select the functions such as permeability, toxic reaction, can make its liver pathological study and drug toxicity test and in terms of Using more accurate.It is constructed simultaneously as artificial liver group is woven on micro-fluidic chip, can easily integrate other tests Module and detection module, so that realizing the correlative study and detection in later period after texture forming with can be convenient.

The above content is combine it is specific/further detailed description of the invention for preferred embodiment, cannot recognize Fixed specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, Without departing from the inventive concept of the premise, some replacements or modifications can also be made to the embodiment that these have been described, And these substitutions or variant all shall be regarded as belonging to protection scope of the present invention.

Claims (13)

1. a kind of artificial liver tissue, which is characterized in that including micro-fluidic chip, the micro-fluidic chip is provided with five layers of knot Structure, including lower die, the first microporous barrier, intermediate chip, the second microporous barrier and the upper die stacked gradually from bottom to top, Be respectively formed down on the lower die, the intermediate chip and the upper die, in, upper three layers of chamber, from bottom to top The culture solution containing growth factor, the extracellular matrix containing hepatic parenchymal cells and hanging containing endothelial cell is perfused in three layers of chamber respectively Liquid is isolated between adjacent chamber with second microporous barrier by first microporous barrier, and the microporous barrier can obstruct cell Travel motion of the flowing of epimatrix without influencing cell passes through the entry and exit feed flow of upper and lower chamber and cultivates, growth because Endothelial cell passes through the extracellular matrix of intermediate cavity and forms lumen under the induction of son, forms the artificial liver containing capillary Tissue.

2. artificial liver tissue as described in claim 1, which is characterized in that the lower die, the intermediate chip and institute Stating upper die is PDMS chip, is assembled in such a way that oxygen plasma is bonded, the microporous barrier passes through itself and the PDMS core The deformation of piece is clamped between the PDMS chip.

3. artificial liver tissue as described in claim 1, which is characterized in that the microporous barrier is that collagen is fabricated to thickness In the film of 0.1 ± 0.05mm range, have aperture in 0.03 ± 0.01mm range, hole distance of shaft centers in 0.06 ± 0.02mm range Microwell array.

4. artificial liver tissue as described in claim 1, which is characterized in that in the entrance integration testing mould of an at least chamber Block integrates detection module in the outlet of an at least chamber, is used for pathological study and drug test.

5. artificial liver tissue as claimed in claim 4, which is characterized in that the test module is containing virus, toxin or medicine The chip of the culture solution of object, the detection module are liver Activity determination module, metabolic integrity detection module or functional production The expression quantity detection module of object.

6. such as artificial liver tissue described in any one of claim 1 to 5, which is characterized in that the lower die, the centre The length × width × height of chip and the upper die is 25mm × 25mm × 1mm；The length × width × height of lower chambers is 5mm × 5mm × 0.2mm, the hole heart of chamber entrance is away from for 18mm；The length × width × height of intermediate cavity is 5mm × 5mm × 1mm, and chamber enters and leaves Mouthful the hole heart away from being 15mm, the intermediate chip the lower die chamber entrance corresponding position equipped with through-hole；Epicoele The length × width × height of room is 5mm × 5mm × 0.2mm, and the hole heart of chamber entrance is away from for 12mm, and the upper die is in described Between chip, the lower die chamber entrance corresponding position be equipped with through-hole；The aperture of each chamber entrance is 1.5mm, respectively Pass through 0.4mm wide between chamber entrance and chamber and the channel contour with chamber is connected to.

7. artificial liver tissue as claimed in claim 6, which is characterized in that all vertical edges in each chamber all pour out radius For the fillet of 1mm, to avoid turbulent flow and bubble is formed；Length × width x thickness of the microporous barrier is 10mm × 10mm × 0.1mm.

8. such as artificial liver tissue described in any one of claim 1 to 5, which is characterized in that use in following configuration at least A kind of: the hepatic parenchymal cells are the cell line HepG2 of the primary hepatic parenchymal cells of people or the hepatic parenchymal cells of people；The cell Epimatrix is gelatinous rat-tail I-type collagen or fibrin, pigskin I-type collagen；The growth factor is Any one or more combination in VEGF, EGF and FGF, the culture solution are the combination of DMEM+RPMI or the group of ECM+DMEM It closes；The endothelial cell is HUVECs or HMEC-1.

9. a kind of production method for making artificial liver tissue as claimed in any one of claims 1 to 8, which is characterized in that packet It includes: making 5 layers of single layer structure of the micro-fluidic chip；Using lower die described in oxygen plasma sputter process lower surface and The upper surface of the intermediate chip, padded on the lower surface of the intermediate chip upper first microporous barrier make its cover chamber and Entrance is not covered, the lower die and the intermediate chip are then aligned by entrance and is made under the lower die Surface and the upper surface of the intermediate chip fasten, and first microporous barrier is clamped wherein；It is fastened using identical operation The lower surface of the upper surface of the intermediate chip and the upper die, and second microporous barrier is clamped wherein；Hereafter The micro-fluidic chip fastened, which is placed on oven for baking, keeps its bonding close.

10. production method as claimed in claim 9, which is characterized in that the microporous barrier is to pass through electrostatic spinning using collagen Method be fabricated to the film of biocompatibility, the method for reusing laser boring processes microwell array on film.

11. the production method as described in claim 9 or 10, which is characterized in that from the entrance of the intermediate cavity in described Between chamber pour into and be uniformly mixed with the extracellular matrixs of hepatic parenchymal cells, filled until in the duct of the outlet of the intermediate cavity, Thereafter the culture solution containing growth factor is injected from the entrance of the lower chambers to lower chambers, from the entrance of the upper chamber to institute Injection endothelial cell suspension in upper chamber is stated, is filled until in the duct of the outlet of the upper chamber, is stood in making for a period of time Simultaneously hereafter adherent growth is continuously injected into culture to the upper chamber from the entrance of the upper chamber on microporous barrier to chrotoplast deposition Liquid；Under the driving of growth factor, endothelial cell vascularization and vertical migration penetrate the extracellular matrix layer of the intermediate cavity, Hereafter the supply for the factor that stops growing is changed to be continuously injected into culture solution, finally contains capillary in intermediate cavity formation Artificial liver tissue.

12. a kind of method using artificial liver tissue as claimed in any one of claims 1 to 8 characterized by comprising To the entrance of chamber injection virus, toxin or drug to be detected, infusion is by the blood vessel of forming into artificial liver tissue Transmitting and diffusion, and generate corresponding reaction；It is detected by the detection module in the exit of chamber.

13. method as claimed in claim 12 characterized by comprising the detection includes liver Activity determination, has been metabolized The expression quantity of whole property detection and functional product detects.